CN114660300A - Method for improving chromatography reagent performance of neocoronal neutralizing antibody by adopting DTT reagent - Google Patents
Method for improving chromatography reagent performance of neocoronal neutralizing antibody by adopting DTT reagent Download PDFInfo
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- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
Abstract
The invention discloses a method for improving the performance of a chromatography reagent of a neocoronal neutralizing antibody by adopting a DTT reagent, which comprises the following steps: preparing an activating solution; preparing a coupling solution; preparing a sealing liquid; preparing a preservation solution; preparing a time-resolved fluorescent microsphere labeled RBD; cleaning for the first time; activating; cleaning for the second time; coupling; cleaning for the third time; and (3) sealing: cleaning for the fourth time; storing; DTT processing time-resolved fluorescent microspheres to mark RBD; preparing a sample pad; preparing a film-scratching coating solution; finally, assembling and slitting are carried out, and then the effect is tested by using a combined card. According to the invention, the proper amount of DTT is adopted to treat time-resolved fluorescent microspheres to mark RBD (radial basis function) to cause microsphere aggregation, so that the method is mild in condition, simple and convenient to operate and good in effect; by improving the detection repeatability of the reagent, a foundation is laid for linear, inter-batch difference and intra-batch difference detection; compared with the traditional ultrasound, the method solves the problem of microsphere aggregation and is beneficial to maintaining the structure and performance of the time-resolved fluorescent microsphere.
Description
Technical Field
The invention relates to the technical field of antibody chromatography reagents, in particular to a method for improving the performance of a chromatography reagent for a neocorona neutralizing antibody by adopting a DTT (diethylenetriamine pentaacetic acid) reagent.
Background
SARS-CoV-2 belongs to beta coronavirus, which is a positive-sense single-stranded RNA virus with envelope. It is transmitted from person to person by droplets or direct contact. Many diagnosed SARS-COV-2 patients develop symptoms of fever or acute respiratory disease (e.g., cough, dyspnea). SARS-CoV-2 has a variety of structural proteins, including spike protein (S), envelope protein (E), membrane protein (M), and nucleocapsid protein (N). The spike protein (S) contains a Receptor Binding Domain (RBD) which is responsible for recognizing the cell surface receptor angiotensin converting enzyme 2(ACE 2). The RBD of the SARS-CoV-2S protein was found to interact strongly with the human ACE2 receptor, leading to endocytosis into the host cell of the deep lung and viral replication. Scientists have demonstrated that the neutralizing epitope is a specific receptor binding domain of the S protein of the viral spike, called S-RBD. After SARS-CoV-2 infection, humans will mount an immune response, including the production of antibodies. Among the total antibodies of the virus, the neutralizing antibody binds to the S-RBD, blocking cellular infiltration and replication of the virus. It is not clear at present how long it takes to produce neutralizing antibodies, and whether they are always produced after SARS-CoV-2 infection or vaccine. Although individuals infected with SARS-CoV-2 will produce antibodies that bind to the virus, not all will produce neutralizing antibodies against SARS-CoV-2.
The reagent for detecting neutralizing antibody in the present market relates to colloidal gold method, chemiluminescence method, enzyme-linked immunization method and fluorescence immunochromatography method. The time-resolved fluorescence immunoassay (TRFIA) is one of the most advanced immunoassay methods in the present generation, combines the amplification effect of a nano signal, has wide application prospect, and is suitable for the development of high-sensitivity immunoassay kits such as tumor markers, hormones and the like.
Different from a classical time-resolved fluorescence immunoassay method DELFIA, the time-resolved fluorescence immunochromatography technology adopts fluorescent nano-microspheres as markers, and each microsphere can be wrapped by thousands of fluorescent molecules, so that the marking efficiency is greatly improved, and the sensitivity is effectively improved; meanwhile, carboxyl with proper density is modified on the surface of the nano fluorescent microsphere for covalent coupling with protein or antibody, so that the stability of the marker is improved.
DTT is a strong reducing agent, the reducing nature of which is largely due to the conformational stability of the six-membered ring (containing the disulfide bonds) in its oxidized state. Its redox potential is-0.33 volts at pH 7. DTT stability is poor due to its easy oxidation by air; but its useful life can be extended by cryopreservation or treatment in an inert atmosphere. As the nucleophilicity of the protonated sulfur is lower, the effective reducibility of DTT decreases with decreasing pH; one of the uses of DTT is as a reducing and deprotecting agent for thiolated DNA. The terminal sulfur atom of thiolated DNA tends to form dimers in solution, particularly in the presence of oxygen. This dimerization greatly reduces the efficiency of some coupling reaction experiments (e.g., the immobilization of DNA in a biosensor); the addition of DTT to a DNA solution, which is removed after a reaction time, can reduce the dimerized DTT of the DNA, and is often used to reduce disulfide bonds in proteins, and to prevent intra-or intermolecular disulfide bonds of proteins formed between cysteines in proteins. However, DTT has previously failed to reduce disulfide bonds embedded within the protein structure (solvent-inaccessible), and reduction of such disulfide bonds often requires first denaturation of the protein (either by heating at high temperature or by addition of a denaturing agent such as 6M guanidine hydrochloride, 8M urea or 1% SDS).
By adopting a competitive inhibition method, the ACE2 recombinant protein and the goat anti-mouse IgG antibody are respectively coated on a membrane detection line and a quality control line. During the detection process, neutralizing antibodies present in the sample will interact with the S-RBD labeled time-resolved fluorescent microspheres and block the protein-protein interaction between S-RBD and ACE 2. When the neutralizing antibody titer in the sample is low or nonexistent, the S-RBD recombinant protein marked by the time-resolved fluorescent microspheres is combined with the ACE2 recombinant protein of the detection line, and a dark band is formed at the detection line. Whether the sample contains SARS-CoV-2 neutralizing antibody or not, the time-resolved fluorescent microsphere labeled quality control antibody (rabbit IgG) is combined with the coating antibody (goat anti-rabbit IgG) on the C line to form a complex and develop color (C line). The concentration of the substance to be detected in the sample can be analyzed by detecting the intensity and the ratio of the fluorescence intensity of the detection line and the fluorescence intensity of the quality control line. The time-resolved fluorescent microspheres have the advantages that: the sensitivity is high and is 2 to 3 orders of magnitude higher than that of gold-labeled and common fluorescence; the quantitative detection can be carried out, and the specific concentration of the object to be detected can be given according to a built-in standard curve; the marker is stable, the anti-interference performance is high, the repeatability of the detection result is good, the operation is simple and convenient, the detection time is short, and the kit can be used for on-site screening; the cost is low, and the cost performance is high. However, based on the problem that the specific sequence of the receptor binding domain (S-RBD) of the virus S protein causes poor repeatability of the reagent, a method for effectively improving the repeatability of the reagent needs to be found out to improve the performance index of the reagent.
Disclosure of Invention
In view of the problems of the prior art, the present invention provides a method for improving the performance of a new crown neutralization antibody chromatography reagent using a DTT reagent.
In order to achieve the above object, the present invention provides a method for improving the performance of a neocorona neutralizing antibody chromatography reagent using a DTT reagent, comprising the steps of:
step S1: preparing an activating solution;
step S2: preparing a coupling solution;
step S3: preparing a sealing liquid;
step S4: preparing a preservation solution;
step S5: preparing a time-resolved fluorescent microsphere labeled RBD;
step S6: labeling RBD with DTT treatment time-resolved fluorescent microspheres: preparing DTT, adding the prepared DTT solution into a time-resolved fluorescent microsphere labeled RBD solution for reduction, centrifuging, supplementing the initial volume with a preservation solution, ultrasonically mixing uniformly, centrifuging, and diluting with a neutralizing antibody diluent;
step S7: preparing a sample pad;
step S8: preparing a film-scratching coating solution;
step S9: and finally, assembling, slitting and testing the effect by using the main card.
Preferably, the step of specifically preparing the activation solution in step S1 is: 0.956 to 0.996g of 2- (N-morpholino) ethanesulfonic acid is selected to be added into 99 to 101ml of Mill-Q purified water, the PH value is adjusted to 6.0 plus or minus 0.1 by NaOH, and then the mixture is stirred and mixed evenly.
Preferably, the coupling solution in step S2 is specifically prepared by the following steps: 0.956 to 0.996g of 2- (N-morpholino) ethanesulfonic acid is added into 99 to 101ml of Mill-Q purified water, the pH value is adjusted to 6.5 +/-0.1 by NaOH, and then the mixture is stirred and mixed evenly.
Preferably, the specific preparation step of the blocking solution in step S3 is: selecting 78-82ml of Mill-Q purified water, 0.95-1.05g of bovine serum albumin, 4.95-5.05g of casein sodium salt, 0.495-0.505ml of 100mM glycine, 0. 200.495-0.505 ml of Tween and 3000.018-0.022 ml of Proclin, mixing and stirring, and finally fixing the volume to 100 ml.
Preferably, the specific preparation step of the preservation solution in step S4 is as follows: selecting 78-82ml of Mill-Q purified water, 0.495-0.505g of bovine serum albumin, 7.95-8.05ml of 100mM glycine, 0.305ml of Tween-200.295 and 3000.018-0.022 ml of Proclin, mixing and stirring, and finally fixing the volume to 100 ml.
Preferably, the preparation of the time-resolved fluorescent microsphere labeled RBD in step S5 specifically includes the steps of: step S501: first cleaning, namely taking an activating solution and time-resolved fluorescent microspheres in a centrifuge tube, then uniformly mixing, centrifuging, supplementing the activating solution to an initial volume, and uniformly dispersing the time-resolved fluorescent microspheres in a buffer solution by using an ultrasonic cleaner;
step S502: activating;
step S503: and (3) cleaning for the second time: centrifuging after the activation is finished, supplementing the volume to the initial volume by using a coupling solution, and ultrasonically mixing uniformly;
step S504: coupling: adding RBD antigen after the second cleaning and mixing uniformly;
step S505, cleaning for the third time, namely centrifuging after coupling is finished, supplementing the initial volume with sealing liquid, and ultrasonically mixing uniformly;
step S506: and (3) sealing:
step S507: fourth cleaning: centrifuging after sealing, supplementing the initial volume with a preservation solution, ultrasonically mixing uniformly, and centrifuging;
step S508: and (5) storing.
Preferably, the activating solution selected in the step S501 is 4 parts, and the time-resolved fluorescent microspheres are 1 part; the activation in step S502 specifically includes: respectively adding the buffer solution washed for the first time in the step S501 into a prepared cross-linking agent A and a prepared cross-linking agent B, wherein the final concentration of the cross-linking agent A is 0.95-1.05mg/ml, and the final concentration of the cross-linking agent B is 1.45-1.55mg/ml, and placing the mixture into a vortex mixer to be mixed uniformly at room temperature; the closing step in the step S506 is to place the solution cleaned for the third time in the step S505 in a vortex mixer for uniform mixing at room temperature; the storage in the step S508 is performed at 2-8 ℃.
Preferably, the specific steps of the reduction in step S6 are: adding 1.9-2.1 μ l of prepared DTT solution into 500 μ l of RBD time-resolved fluorescent microsphere solution, and respectively reducing in a room temperature (48-52) rpm rotary mixer for 30min, 45min and 60 min; the specific steps of diluting with the neutralizing antibody diluent in the step S6 are as follows: directly marking the RBD by the time-resolved fluorescent microspheres and diluting the RBD by the well-reduced time-resolved fluorescent microspheres with neutralizing antibody diluent to three different gradients, then carrying out spray-pad treatment on the RBD with the solubility of 0.1mg/ml, and then carrying out strip cutting assembly to test the effect after drying.
Preferably, the sample pad in step S7 is prepared by the following steps: selecting 78-82ml of Mill-Q purified water, 4.95-5.05g of casein, 1.98-2.02g of trehalose, 6.00-6.10g of Tween-207.98-8.02ml of Tris and 200000.95-1.05 g of PEG, mixing and stirring, adjusting the pH to 8.5 +/-0.1, finally setting the volume to 1000ml, spraying 50ml of prepared sample pad solution on 6613, drying, and cutting into 17mm wide for later use.
Preferably, the step S8 of preparing the coating solution for scratching the film specifically comprises the following steps: 24.9-25.1ml of trehalose 20%, 4.98-5.02ml of methanol and 79.5-80.5ml of 1 XPBS are selected to be mixed and stirred to prepare a film-scribing coating solution, the prepared film-scribing coating solution is diluted with ACE2 to 1.0mg/ml for film-scribing coating, and then the film-scribing coating solution is dried for standby.
By adopting the technical scheme of the invention, the invention has the following beneficial effects:
1. the method adopts proper DTT processing time to distinguish the phenomenon of microsphere aggregation caused by fluorescence labeling RBD, and is a mode with mild conditions, simple and convenient operation and good effect.
2. By improving the detection repeatability of the reagent, a foundation is laid for linear, inter-batch difference and intra-batch difference detection.
3. Compared with the traditional ultrasound, the method solves the problem of microsphere aggregation and is beneficial to maintaining the structure and performance of the time-resolved fluorescent microsphere.
Detailed Description
The present invention will be further described with reference to the following examples.
In order to achieve the above object, the present invention provides a method for improving the performance of a neocorona neutralizing antibody chromatography reagent using a DTT reagent, comprising the steps of:
step S1: preparing an activating solution (solution A);
step S2: preparing a coupling solution (solution C);
step S3: preparing sealing liquid (S liquid);
step S4: preparing a preservation solution (P solution);
step S5: configuring time-resolved fluorescent microsphere mark RBD;
step S501: first cleaning, namely taking the activating solution (solution A) and the time-resolved fluorescent microspheres in a centrifuge tube, then uniformly mixing, centrifuging (centrifuging for 15min under the conditions of 4 ℃ and 15000 g), supplementing the initial volume with the solution A (pH 6.0), and uniformly dispersing the time-resolved fluorescent microspheres in a buffer solution by using an ultrasonic cleaner;
step S502: activating;
step S503: and (3) cleaning for the second time: centrifuging (centrifuging at 4 deg.C under 15000g for 15min) after activation, removing supernatant, supplementing to initial volume with coupling solution (solution C), and mixing with ultrasound;
step S504: coupling: adding RBD antigen after the second cleaning and mixing uniformly;
step S505, cleaning for the third time, namely centrifuging (centrifuging for 15min at the temperature of 4 ℃ under the condition of 15000 g) after the coupling is finished, removing supernatant, supplementing the supernatant to the initial volume by using confining liquid (S liquid), and ultrasonically mixing the confining liquid and the S liquid;
step S506: and (3) sealing:
step S507: and (3) fourth cleaning: centrifuging (at 4 deg.C under 15000g for 15min), removing supernatant, supplementing to initial volume with preservation solution (P solution), ultrasonically mixing, and centrifuging;
step S508: storing;
step S6: labeling RBD with DTT treatment time-resolved fluorescent microspheres: preparing DTT, adding the prepared DTT solution into RBD time-resolved fluorescent microsphere solution for reduction, centrifuging (centrifuging for 15min at 4 ℃ under the 15000g factor), supplementing the initial volume with preservation solution P, ultrasonically mixing, centrifuging, and diluting with neutralizing antibody diluent;
step S7: preparing a sample pad;
step S8: preparing a film-scratching coating solution;
step S9: and finally, assembling, slitting and testing the effect by using a combined card.
The step of specifically preparing the activating solution (solution a) in step S1 is: 0.976g of 2- (N-morpholino) ethanesulfonic acid is added into 100ml of Mill-Q purified water, the pH value is adjusted to 6.0 +/-0.1 by NaOH, and then stirring and mixing are carried out.
The coupling solution (solution C) in step S2 is specifically prepared by the steps of: 0.976g of 2- (N-morpholino) ethanesulfonic acid was added to 100ml of Mill-Q purified water, adjusted to pH 6.5. + -. 0.1 with NaOH, and then stirred and mixed.
Referring to table 1 below, the blocking solution (S solution) in step S3 is specifically prepared by the following steps: selecting and mixing 80ml of Mill-Q purified water, 1g of bovine serum albumin, 5g of casein sodium salt, 0.5ml of 100mM glycine, 200.5 ml of Tween-and 3000.02ml of Proclin, stirring, and finally fixing the volume to 100 ml.
TABLE 1
Referring to table 2 below, the specific preparation steps of the preservation solution (solution P) in step S4 are as follows: selecting and mixing 80ml of Mill-Q purified water, 0.5g of bovine serum albumin, 8ml of 100mM glycine, 200.3 ml of Tween-and 3000.02ml of Proclin, stirring, and finally fixing the volume to 100 ml.
TABLE 2
The activating solution (solution A) selected in the step S501 is 4 parts, and the time-resolved fluorescent microspheres are 1 part.
The activation in step S502 specifically includes: respectively adding the buffer solution washed for the first time in the step S501 into a prepared cross-linking agent A and a prepared cross-linking agent B, wherein the final concentration of the cross-linking agent A is 1.0mg/ml, and the final concentration of the cross-linking agent B is 1.5mg/ml, and placing the mixture in a vortex mixer to mix uniformly for 20min at room temperature;
the final concentration in step S504 was 0.2mg/ml, and the mixture was placed in a vortex mixer and mixed for 2.5h at room temperature.
The closing step in the step S506 is specifically that the solution cleaned for the third time in the step S505 is placed in a vortex mixer and mixed uniformly for 1.5 hours at room temperature; the storage in the step S508 is performed at 2-8 ℃.
The specific preparation of the DTT in step S6 includes: 0.75mg, 1.0mg, 1.25mg, 1.50mg DTT were added to 20ml of Mill-Q purified water, and vortexed to give respective concentrations of: 37.5ug/ml, 50ug/ml, 62.5ug/ml, 75 ug/ml.
The specific steps of the reduction in the step S6 are as follows: and (3) respectively adding 2 mu l of the prepared DTT solution into 500 mu l of RBD-marked time-resolved fluorescent microsphere solution, and respectively reducing in a room-temperature 50rpm rotary mixer for 30min, 45min and 60 min. The specific steps of diluting with the neutralizing antibody diluent in the step S6 are as follows: directly marking the RBD by the time-resolved fluorescent microspheres and marking the RBD by the reduced time-resolved fluorescent microspheres by using neutralizing antibody diluent to dilute three different gradients, then carrying out spray-pad treatment on the RBD with the solubility of 0.1mg/ml, and carrying out strip cutting (17mm) assembly test on the RBD after drying for 24 hours at the temperature of 45 ℃.
Referring to table 3, the specific steps of preparing the sample pad in step S7 are as follows: selecting and mixing 80ml of Mill-Q purified water, 5g of casein, 2g of trehalose, Tween-208 ml, 6.05g of Tris and PEG 200001 g, stirring, adjusting the pH value to 8.5, finally fixing the volume to 1000ml, spraying 50ml of prepared sample pad solution on 6613, drying at 45 ℃ for 24 hours, and cutting into 17mm wide for later use.
TABLE 3
Referring to table 4, the step S8 of preparing the coating solution for scratching the film specifically includes: 25ml of trehalose 20%, 5ml of methanol and 80ml of 1 XBS are selected to be mixed and stirred to prepare a film-scratching coating solution, the prepared film-scratching coating solution is diluted with ACE2 to 1.0mg/ml, film-scratching coating is carried out, and the film-scratching coating solution is dried at the temperature of 45 ℃ for 48 hours for later use.
TABLE 4
And the assembly substances in the step S9 comprise a prepared sample pad, a RBD (radial basis function) fluorescent pad, an ACE2 NC (acrylonitrile-butadiene-styrene) film and absorbent paper.
The chromatography reagent prepared by the formula is used for testing internal reference substances, and the performance index of the reagent is evaluated.
Table 1-a is: and (3) directly marking the RBD spray pad by using the time-resolved fluorescent microspheres to obtain a repeatability experiment result.
TABLE 1-a
Table 1-b time resolved fluorescent microspheres directly labeled RBD spray pad inter-batch difference experimental results.
TABLE 1-b
Table 1-c time resolved fluorescent microspheres directly labeled RBD spray pad linear experimental results.
TABLE 1-c
TABLE 2-a DTT (37.5ug/ml DTT, 30min reduction) reduction time resolved fluorescent microspheres directly labeled RBD spray pad repeatability experiment results.
TABLE 2-a
Table 2-b DTT (37.5ug/ml DTT, 30min reduction) reduction time resolved fluorescent microspheres directly labeled RBD inter-batch difference experimental results.
TABLE 2-b
Table 2-c DTT (37.5ug/ml DTT, 30min reduction) reduction time resolved fluorescent microspheres directly labeled RBD linear experiment results.
TABLE 2-c
Table 3-a repeated experimental results of RBD directly marked by DTT (50ug/ml DTT, 30min reduction) reduction time resolution fluorescent microspheres.
TABLE 3-a
Table 3-b DTT (50ug/ml DTT, 30min reduction) reduction time resolved fluorescent microspheres directly labeled RBD inter-batch difference experimental results.
TABLE 3-b
Table 3-c DTT (50ug/ml DTT, 30min reduction) reduction time resolved fluorescent microspheres directly labeled RBD linear experiment results.
TABLE 3-c
TABLE 4-a DTT (62.5ug/ml DTT, 30min reduction) reduction time resolved fluorescent microspheres directly labeled RBD repeatability experiment results.
TABLE 4-a
Table 4-b DTT (62.5ug/ml DTT, 30min reduction) reduction time resolved fluorescent microspheres directly labeled RBD inter-batch difference experimental results.
TABLE 4-b
Table 4-c DTT (62.5ug/ml DTT, 30min reduction) reduction time resolved fluorescent microspheres directly labeled RBD linear experiment results.
TABLE 4-c
TABLE 5-a DTT (75ug/ml DTT, reduction 30min) reduction time resolved fluorescent microspheres directly labeled RBD repeatability experiment results.
TABLE 5-a
Table 5-b DTT (75ug/ml DTT, 30min reduction) reduction time resolved fluorescent microspheres directly labeled RBD inter-batch difference experimental results.
TABLE 5-b
Table 5-c DTT (75ug/ml DTT, 30min reduction) reduction time resolved fluorescent microspheres directly labeled RBD linear experiment results.
TABLE 5-c
TABLE 6-a DTT (37.5ug/ml DTT, reduction 45min) reduction time resolved fluorescent microspheres directly labeled RBD repeatability experiment results.
TABLE 6-a
Table 6-b DTT (37.5ug/ml DTT, reduction 45min) reduction time resolved fluorescent microspheres directly labeled RBD inter-batch difference experimental results.
TABLE 6-b
Table 6-c DTT (37.5ug/ml DTT, reduction 45min) reduction time resolved fluorescent microspheres directly labeled RBD linear experimental results.
TABLE 6-c
TABLE 7-a DTT (50ug/ml DTT, reduction 45min) reduction time resolved fluorescent microspheres directly labeled RBD repeatability experiment results.
TABLE 7-a
Table 7-b DTT (50ug/ml DTT, reduction 45min) reduction time resolved fluorescent microspheres directly labeled RBD inter-batch difference experimental results.
TABLE 7-b
Table 7-c DTT (50ug/ml DTT, reduction 45min) reduction time resolved fluorescent microspheres directly labeled RBD linear experiment results.
TABLE 7-c
TABLE 8-a DTT (62.5ug/ml DTT, reduction 45min) reduction time resolved fluorescent microspheres directly labeled RBD repeatability experiment results.
TABLE 8-a
Table 8-b DTT (62.5ug/ml DTT, reduction 45min) reduction time resolved fluorescent microspheres directly labeled RBD inter-batch difference experimental results.
TABLE 8-b
TABLE 8-c DTT (62.5ug/ml DTT, reduction 45min) reduction time resolved fluorescent microspheres directly labeled RBD linear experimental results.
TABLE 8-c
TABLE 9-a DTT (75ug/ml DTT, reduction 45min) reduction time resolved fluorescent microspheres directly labeled RBD repeated experimental results.
TABLE 9-a
Table 9-b DTT (75ug/ml DTT, reduction 45min) reduction time resolved fluorescent microspheres directly labeled RBD inter-batch difference experimental results.
TABLE 9-b
Table 9-c DTT (75ug/ml DTT, reduction 45min) reduction time resolved fluorescent microspheres directly labeled RBD linear experiment results.
TABLE 9-c
TABLE 10-a DTT (37.5ug/ml DTT, reduction 60min) reduction time resolved fluorescent microspheres directly labeled RBD repeated experimental results.
TABLE 10-a
Table 10-b DTT (37.5ug/ml DTT, reduction 60min) reduction time resolved fluorescent microspheres directly labeled RBD inter-batch difference experimental results.
TABLE 10-b
Table 10-c DTT (37.5ug/ml DTT, 60min reduction) reduction time resolved fluorescent microspheres directly labeled RBD linear experimental results.
TABLE 10-c
Table 11-a (50ug/ml DTT, reduction 60min) DTT reduction time resolved fluorescent microspheres directly labeled RBD repeated experimental results.
TABLE 11-a
Table 11-b DTT (50ug/ml DTT, reduction 60min) reduction time resolved fluorescent microspheres directly labeled RBD inter-batch difference experimental results.
TABLE 11-b
Table 11-c DTT (50ug/ml DTT, reduction 60min) reduction time resolved fluorescent microspheres directly labeled RBD linear experimental results.
TABLE 11-c
TABLE 12-a DTT (62.5ug/ml DTT, reduction 60min) reduction time resolved fluorescent microspheres directly labeled RBD repeated experimental results.
TABLE 12-a
Table 12-b DTT (62.5ug/ml DTT, reduction 60min) reduction time resolved fluorescent microspheres directly labeled RBD inter-batch difference experimental results.
TABLE 12-b
Table 12-c DTT (62.5ug/ml DTT, 60min reduction) reduction time resolved fluorescent microspheres directly labeled RBD linear experiment results.
TABLE 12-c
Table 13-a DTT (75ug/ml DTT, reduction 60min) reduction time resolved fluorescent microspheres directly labeled RBD repeatability experiment results.
TABLE 13-a
Table 13-b DTT (75ug/ml DTT, reduction 60min) reduction time resolved fluorescent microspheres directly labeled RBD inter-batch difference experimental results.
TABLE 13-b
Table 13-c DTT (75ug/ml DTT, reduction 60min) reduction time resolved fluorescent microspheres directly labeled RBD linear experimental results.
TABLE 13-c
Analysis from the above experimental results: the performance evaluation experiment result of the chromatographic reagent shows that the RBD marked by using the DTT reagent to reduce the time-resolved fluorescent microspheres obviously improves the performance indexes of the reagent, and comprises the following steps: repeatability, batch-to-batch difference, optimal reduction conditions obtained by screening the DTT dosage and the reduction time are as follows: the concentration of DTT (62.5ug/ml DTT, reduction 60min), the repeatability of the internal reference (0.31ug/ml, 1.25ug/ml) tested at this time was 7.11% and 7.52% respectively; the test internal reference (0.31ug/ml, 1.25ug/ml) had batch to batch differences of 9.03%, 8.45%, respectively; internal references (0.12ug/ml, 0.69ug/ml, 1.39ug/ml, 2.08ug/ml, 2.77ug/ml, 3.47ug/ml, 4.16ug/ml) were tested for linear equations: y is 0.6436x-0.7229, R2 is 0.9917, the method is linear in the concentration range of 0.12-4.16 ug/ml.
The invention is a phenomenon that the aggregation of time-resolved fluorescent microspheres is caused by reducing a disulfide bond formed by cysteine in RBD marked on the time-resolved fluorescent marked microspheres by using a DTT reagent, and the invention has the advantages that:
1. because the invention adopts a proper amount of DTT to reduce the RBD marked by the time-resolved fluorescent microspheres, the reaction condition is mild, and the structure of the antigen S-RBD can not be damaged, thereby solving the problem that the aggregation of the time-resolved microspheres is changed by utilizing physical characteristics such as ultrasonic (the structure and the performance of the time-resolved fluorescent microspheres can be changed due to long ultrasonic time).
2. By improving the aggregation problem of the time-resolved fluorescent microspheres, the invention improves a series of performance indexes of the detection reagent, such as repeatability, inter-batch difference, intra-batch difference, linearity and the like.
The above description is only a preferred embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications and equivalents of the present invention, which are made by the present specification and directly/indirectly applied to other related technical fields within the spirit of the present invention are included in the scope of the present invention.
Claims (10)
1. A method for improving the performance of a chromatography reagent for a neocorona neutralizing antibody by using a DTT reagent, comprising the steps of:
step S1: preparing an activating solution;
step S2: preparing a coupling solution;
step S3: preparing a sealing liquid;
step S4: preparing a preservation solution;
step S5: preparing a time-resolved fluorescent microsphere labeled RBD;
step S6: labeling RBD with DTT treatment time-resolved fluorescent microspheres: preparing DTT, adding the prepared DTT solution into a time-resolved fluorescent microsphere labeled RBD solution for reduction, centrifuging, supplementing the initial volume with a preservation solution, ultrasonically mixing uniformly, centrifuging, and diluting with a new corona neutralizing antibody diluent;
step S7: preparing a sample pad;
step S8: preparing a film-scratching coating solution;
step S9: and finally, assembling, slitting and testing the effect by using a combined card.
2. The method for improving the performance of a chromatography reagent for neocoronal neutralizing antibodies using DTT reagent of claim 1, wherein the step of formulating the activating solution in step S1 comprises the steps of: 0.956 to 0.996g of 2- (N-morpholino) ethanesulfonic acid is selected to be added into 99 to 101ml of Mill-Q purified water, the PH value is adjusted to 6.0 plus or minus 0.1 by NaOH, and then the mixture is stirred and mixed evenly.
3. The method for improving the performance of a chromatography reagent for neocorona-neutralizing antibodies by using DTT reagent as claimed in claim 1, wherein the step of formulating the coupling solution in step S2 is: 0.956 to 0.996g of 2- (N-morpholino) ethanesulfonic acid is added into 99 to 101ml of Mill-Q purified water, the pH value is adjusted to 6.5 +/-0.1 by NaOH, and then the mixture is stirred and mixed evenly.
4. The method for improving the performance of a chromatography reagent for neocoronal neutralizing antibodies by using DTT reagent of claim 1, wherein the blocking solution of step S3 is prepared by the following steps: selecting 78-82ml of Mill-Q purified water, 0.95-1.05g of bovine serum albumin, 4.95-5.05g of casein sodium salt, 0.495-0.505ml of 100mM glycine, 0. 200.495-0.505 ml of Tween and 3000.018-0.022 ml of Proclin, mixing and stirring, and finally fixing the volume to 100 ml.
5. The method for improving performance of a chromatography reagent for a neocorona neutralizing antibody by using a DTT reagent as claimed in claim 1, wherein the specific preparation steps of the preservation solution in the step S4 are as follows: selecting 78-82ml of Mill-Q purified water, 0.495-0.505g of bovine serum albumin, 7.95-8.05ml of 100mM glycine, 0.305ml of Tween-200.295 and 3000.018-0.022 ml of Proclin, mixing and stirring, and finally fixing the volume to 100 ml.
6. The method for improving the performance of the chromatography reagent for neocoronal neutralizing antibodies using the DTT reagent of claim 1, wherein the step S5 of preparing the time-resolved fluorescence microsphere labeled RBD comprises the following steps:
step S501: a first cleaning step, namely, taking the activation liquid and the time-resolved fluorescent microspheres in a centrifuge tube, then uniformly mixing, centrifuging, supplementing the activation liquid to an initial volume, and uniformly dispersing the time-resolved fluorescent microspheres in a buffer solution by using an ultrasonic cleaner;
step S502: activating;
step S503: and (3) cleaning for the second time: centrifuging after the activation is finished, supplementing the volume to the initial volume by using a coupling solution, and ultrasonically mixing uniformly;
step S504: coupling: adding RBD antigen after the second cleaning and mixing uniformly;
step S505, cleaning for the third time, namely centrifuging after coupling is finished, supplementing the initial volume with confining liquid, and then ultrasonically mixing uniformly;
step S506: and (3) sealing:
step S507: fourth cleaning: centrifuging after sealing, supplementing the volume to the initial volume with a preservation solution, ultrasonically mixing uniformly, and centrifuging;
step S508: and (5) storing.
7. The method for improving the performance of the chromatography reagent for neocoronal neutralizing antibodies by using the DTT reagent of claim 6, wherein the amount of the activating solution selected in the step S501 is 4, and the amount of the time-resolved fluorescent microspheres is 1; the activation in step S502 specifically includes: respectively adding the buffer solution washed for the first time in the step S501 into a prepared cross-linking agent A and a prepared cross-linking agent B, wherein the final concentration of the cross-linking agent A is 0.95-1.05mg/ml, and the final concentration of the cross-linking agent B is 1.45-1.55mg/ml, and placing the cross-linking agent A and the cross-linking agent B into a vortex mixer for uniformly mixing at room temperature; the closing step in the step S506 is to place the solution cleaned for the third time in the step S505 in a vortex mixer for uniform mixing at room temperature; the storage in the step S508 is performed at 2-8 ℃.
8. The method for improving the performance of a chromatography reagent for neocorona neutralizing antibodies by using DTT reagent as claimed in claim 1, wherein the specific steps of reduction in step S6 are: adding 1.9-2.1 μ l of prepared DTT solution into 500 μ l of RBD time-resolved fluorescent microsphere solution, and reducing in a rotary mixer at room temperature of 48-52rpm for 30min, 45min and 60 min; the specific steps of diluting with the neutralizing antibody diluent in the step S6 are as follows: directly marking the time-resolved fluorescent microspheres with RBD and diluting the reduced time-resolved fluorescent microspheres with RBD by using neutralizing antibody diluent, then carrying out spray-pad treatment on the mixture with the solubility of 0.1mg/ml, and then carrying out slitting assembly after drying to test the effect.
9. The method for improving the performance of a chromatography reagent for neocorona neutralizing antibodies by using DTT reagent as claimed in claim 1, wherein the sample pad of step S7 is prepared by the following steps: selecting 78-82ml of Mill-Q purified water, 4.95-5.05g of casein, 1.98-2.02g of trehalose, 6.00-6.10g of Tween-207.98-8.02ml of Tris and 200000.95-1.05 g of PEG, mixing and stirring, adjusting the pH to 8.5 +/-0.1, finally fixing the volume to 1000ml, spraying 50ml of prepared sample pad solution on 6613, drying, and cutting into 17mm wide for later use.
10. The method for improving the performance of the chromatography reagent for the neocorona neutralizing antibody by using the DTT reagent as claimed in claim 1, wherein the step S8 of preparing the membrane-dividing coating solution comprises the following steps: 24.9-25.1ml of trehalose 20%, 4.98-5.02ml of methanol and 79.5-80.5ml of 1 XPBS are selected to be mixed and stirred to prepare a film-scratching coating solution, the prepared film-scratching coating solution is diluted with ACE2 to 1.0mg/ml for film-scratching coating, and then the film-scratching coating solution is dried for standby.
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